Indonesian Journal of Cancer Chemoprevention, 2011, 2(2):233-240

ISSN : 2088-0197123

233
Activity of Trigonella foenum-graecum
on Some Cell Lines.


Kurnia Agustini, Wahono Sumaryono, R. Micho Widyanto

Center for Pharmaceuticals and Medical Technology
Laboratory for Development of Industrial Agro and Biomedical Technology
Agency for the Assessment and Application of Technology ( BPPT)
Kawasan PUSPIPTEK, Serpong Indonesia



ABSTRACT

Trigonella foenum-graecum (TFG) is one of medicinal plants contains some
steroidal sapogenin such as diosgenin, yamogenin, gitogenin, tigogenin and trigoneoside,
also alkaloid trigonellin, which is have many activity as antidiabetic, estrogenic and also
as anti cancer. This experiment was done to explore the activity of some extract of
TFG on some cell lines such as MCF7 (Human Breast Cancer Cell-line), T47D (Human
Breast Cancer Cell-line), PC3 (Human Prostate Cell-line) and SKOV (Human Ovarian
Carcinoma Cell-line). This assay was done using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium) methods. Results showed that ethyl acetate fraction gives the
lowest IC50 than another extracts. IC50 for PC3 is 66.24 ppm, IC50 for MCF7 is 41.81
ppm, IC50 for T47D is 58.63 ppm. These datas can be used for further research to
isolate the active compound from TFG.

Keywords: Trigonella foenum-graecum, MCF-7, T47D, SKOV, PC3.


INTRODUCTION
Fenugreek seed or Foenigraeci
semen is dried seed from Trigonella foenum-
graecum L., Leguminosae, (Anonim, 1979).
Geographical distribution of Fenugreek is
indigenous to the Mediterranean region,
China, India and Indonesia. Fenugreek
contains alkaloids (trigonelline, an alkaloid
pyridine, gentianin and karpain), flavonoids
e.g. vitexin in glycoside or ester form,
isovitexin, orientin, vicenin, quercetin and
luteolin, some sapogenin steroid ingredients,
e.g. diosgenin, precursor for sexual hormone
(Evans, 2002), its isomer Yamogenin
(Dewick, 1997), gitogenin, tigogenin, and
trigoneoside (saponine like estrogen) which
have effect as phytoestrogen for menopause
symptoms therapy. Fenugreek contains
diosgenin in base free form 0.8 2.2 %.
Fenugreek also contains fatty oil 20-30%,
essential oil, saponine, nicotinamide, choline,
bitter

compound and mucilage (Evans, 2002).
Empirically fenugreek was use as
aphrodisiac, carminative, diuretic,
emmenagogue, emollient, galactogogue and
tonic. Many researches, preclinically and
clinically, showed that fenugreek have
activity for diabetic disease (Annida and
Stanley 2004). Fenugreek also have
estrogenic effect, as Phytoestrogen, predicted
by its sapogenin steroid contains (Agustini,
2007). It can induce uterine contraction, so it
should be avoided during pregnancy.
Phytoestrogen is used as alternative for
Hormone Replacement Therapy (HRT) to
help reducing menopause symptoms. It can
be used for long term until the body can make
adaptation on the new level hormone
(Badziad, 2003).


*Corresponding author email : kurnia_atini@yahoo.com
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234




Figure 1. Some chemicals contains of Trigonella foenum-graecum L. Diosgenin (A), Yamogenin (B),
Protodioscin (C), and Tigogenin (D).

Medicinal plants have been widely
used for cancer treatment both as prevention
and treatment. In contrast to cancer treatment
using synthetic drug that can be given as a
primary drug or as an additional therapy
(adjuvant), treatment with medicines derived
from plants can also be intended for
prevention (chemopreventive). The aim of
treatment is approximately similar by
synthetic drugs such as chemotherapy;
immunotherapy or palliative therapies and
cancer pain, in practice, always use the
combination therapy treatment of several
kinds of medicinal plants by also predicting
the side effects that may occur (Maat, 2003).
Cancer is a malignant neoplasm and
a growing network of small pieces that
become large and unmanageable. To date
approximately 120 types of cancer are known
and classified in 12 major parts, namely:
uterine cancer, breast, respiratory system,
digestive organs, bones and muscles, urinary
tract, skin, lymph, blood, eyes,
gastrointestinal tract and nervous system
(Saputra et al., 2000). Cancer treatment is
medically requiring a very high cost.
Clinically, the common cancer treatments are
surgery, radiation and chemotherapy. In
addition to these conventional treatments,
also develop treatment using natural
ingredients.
Screening for anticancer from
medicinal plants has already done for many
years ago in many research institute in the
world, and sometimes it takes a long time
research. Meanwhile, many medicinal plants
which empirically has been used but have no
scientific data. Cytotoxicity test using human
or animal cell-lines are often used as a
preliminary test in order to search for new
cancer drugs. Antiviral and anticancer drug is
a compound inhibiting cell growth without
damaging the normal cells. According to the
definition of National Cancer Institute (NCI)
cytotoxicity test is only a test of toxicity to
cells (tumors) in the media. Screen of the next
antitumor or antineoplastic should be done by
in vivo experiment. The term of anti cancer
can be used when the results of clinical trials
in human already done (Scheuer, 1987).
Refer to data that showed TFG
having estrogenic activity as phytoestrogen,
inspiring to study its possibility as Selective
Estrogen Receptor Modulators (SERMs)
candidate. SERMs such as tamoxifen, which
are used clinically for the treatment of breast
cancer, act as estrogen agonists in certain
tissues but exhibit antiestrogenic effects in
others (Rosenbaum and Osborne, 2000).
There are several researches about SERMs
candidates for post tumor surgery therapy
from phytoestrogen pure compounds, such as
C
D
A B
Agustini et al.
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235
dammarane, a sapogenine steroid from
ginseng (Oh et al., 1999), Genistein (an
isoflavone from soybean) and resveratrol, a
stilbene from grape (Baht et al., 2001).

METHODS
Samples Preparation
TFG seeds were obtained from
Tawangmangu, Central Java, Indonesia.
Seeds were dried and grind, then were
extracted with methanol and ethanol. The
methanolic extract was fractioned with n-
hexane, ethylacetic (EtOAc) and n-buthanol.
Every extract and fraction was dried with
vacuum rotary evaporator.

Cell Culture
The cell lines PC3 (Human Prostate
Cancer), SKOV3 (Human Ovarian
Carcinoma), MCF-7 (Human Breast
Adenocarcinoma), T47D (Human Breast
Cancer) were obtained from Laboratory for
Development of Industrial Agro and
Biomedical Technology (LAPTIAB-BPPT)
Indonesia. Cells were routinely maintained
and grown in 75 cm
2
flasks at 37
0
C, 5% CO
2

and in a 95% humidified atmosphere. The
growth medium was prepared as following :
RPMI 1640,Gibco life Technologies with
phenol red and 2 mM glutamine, 100 U/ml
penicillin, 0.1 mg/ml Streptomycin, 1 mM
sodium pyruvate and supplemented with 10%
Foetal Bovine Serum (FBS, Gibco Life
Technologies) which already heat inactivated
at 56
0
C for 30 min. Passaging of cells was
carried out using 4 ml of trypsin-EDTA at
room temperature for 75 cm
2
flask,
respectively for 3 min. After that, 10 ml
media with 10% FBS were used to reduce the
action of trypsin on cells. After
centrifugation, the obtained cells were
platted.

Cytotoxicity Test with MTT Method
Cells were platted into 96-well plates
(10,000 cells/well) in medium RPMI with
phenol red containing 10%Fetal Bovine
Serum (FBS), 100U/ml penicillin, 0.1 mg/ml
streptomycin and 1mM sodium pyruvate,
then incubated for 24 hours at 37
0
C, 5% CO
2

and in a 95% humidified atmosphere. After
24 hours, medium was changed with samples
(extracts and phases of TFG) in growth
medium in different concentration and
incubated for another 24 hours at 37
0
C, 5%
CO
2
and in a 95% humidified atmosphere.
Assays were done in wide range
concentration, from 10 ppm until 500 ppm,
divide into six variation concentration.
After 24 hours treatment, the cells
were washed with Phosphate Buffer Saline
(PBS). Then the MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium) solution in medium,
was added followed by incubation for 4 hours
at 37
0
C, 5% CO
2
and in a 95% humidified
atmosphere. The crystal of formazan blue
will be formed. After that, reaction was
stopped by added Sodium Dodecyl Sulphate
(SDS) into every well. Leave plate in dark
place for 12 hours (overnight). The intensity
of the color formed was measured by ELISA
reader at 570nm.

RESULTS
Results of percentage growth of
SKOV cells, MCF7 cells, T47D cells and
PC3 cells were performed in table I table IV
and figure 2-5. Each data was presented from
6 (six) concentration variation, with n=3.

Table I. Percentage growth of Trigonella foenum-graecum on SKOV3 cells (n=3)

Concentration
(ppm)
% Growth SKOV3 cells
EtOH extr. MetOH extr. EtOAc Phase
10 106.36 96.87 102.80
20 106.04 96.17 96.28
50 89.97 77.08 96.33
100 97.09 58.31 96.87
250 42.50 20.44 28.53
500 12.24 12.24 12.89

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236

Figure 2. Effect of TFG on growth of SKOV3 cells. The results were plotted as percent of SKOV3
growth (relative cell growth (%)) versus concentration part permillion (ppm) (n=3)


Table II. Percentage growth of Trigonella foenum-graecum on MCF7 cells (n=3)
Concentration
(ppm)
%Growth MCF7 cells
MetOH extr. EtOH extr. EtOAc Phase Hexan Phase BuOH Phase Water Phase
10 110.61 116.21 78.72 75.17 107.74 100.89
20 111.21 111.33 78.54 92.71 97.80 107.95
50 98.67 119.53 30.20 82.82 73.13 102.25
100 46.65 67.57 19.47 46.90 32.25 100.05
250 18.57 18.63 17.96 18.99 20.54 92.21
500 17.06 17.60 17.90 18.87 18.82 73.34



Figure 3. Effect of TFG on growth of MCF7 cells. The results were plotted as percent of MCF7
growth (relative cell growth (%)) versus concentration part permillion (ppm) (n=3)





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237


Table III. Percentage growth of Trigonella foenum graecum on T47D cells (n=3)

Concentration
(ppm)
% Growth T47D cells
MetOH extr. EtOH extr. EtOAc Phase Hexane Phase
10 102.28 112.25 88.42 96.75
20 68.69 68.27 90.92 98.94
50 92.78 78.58 33.50 111.20
100 55.81 54.25 17.28 30.21
250 16.69 16.73 16.65 16.35
500 15.72 16.05 16.86 16.69



Figure 4. Effect of TFG on growth of T47D cells. The results were plotted as percent of T47D
growth (relative cell growth (%)) versus concentration part permillion (ppm)


Table IV. Percentage growth of Trigonella foenum graecum on PC3 cells (n=3)

Concentration
(ppm)
%Growth PC3 cells
EtOH extr. MetOH extr. EtOAc Phase
10 93.44 87.79 75.39
20 93.93 80.20 70.65
50 61.66 89.15 59.65
100 57.41 59.04 58.02
250 16.15 14.60 20.48
500 16.27 14.52 15.05

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238

Figure 5. Effect of TFG on growth of PC3 cells. The results were plotted as percent of PC3 growth
(relative cell growth (%)) versus concentration part permillion (ppm)

Tables V. IC50 value from some extracts and phases of TFG on PC3, SKOV3, MCF7 and T47D cell-
lines. Each data was obtained from six variation concentration with n=3

No. Cell-line Samples of TFG IC
50
(ppm)
1.
PC-3

EtOH Extract 101.52
2. MetOH Extract 99.05
3. EtOAc Phase 66.24
4.
SKOV3
EtOH Extract 230.92
5. MetOH Extract 116.48
6. EtOAc Phase 208.74
7.


MCF7
MetOH Extract. 186.09
8. EtOH Extract 241.24
9. EtOAc Phase 41.81
10. Hexane Phase 102.11
11. BuOH Phase 136.25
12. Water Phase 1569.26
13.
T47D

MetOH Extract 140.31
14. EtOH Extract 128.37
15. EtOAc Phase 58.63
16. Hexane Phase 206.60


DISCUSSION
Recent studies suggest that TFG and
its active compounds may possess
anticarcinogenic potential. Raju et al. (2004)
showed that diosgenin, a steroid saponin from
TFG can inhibit azoxymethane-induced
aberrant crypt foci formation in F344 rats and
induces apoptosis in HT-29 human colon
cancer cells. Also refer from Agustini et al.
(2007) that showed that TFG have estrogenic
effect on ovariectomized and immature rats.


Some phytoestrogens are believed to have
selective estrogen receptor modulator
(SERM) activity with no action in the uterus
but beneficial effects in the
hypothalamus/pituitary unit and in the bone
and are presently the focus of clinical interest
(Wuttke et al., 2003). So it is an interesting
phenomena to investigate and screen TFG
potency on some cancer cell line, especially
cell line with estrogen receptor positive.
Agustini et al.
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239
Cell lines used in this assay are PC3,
SKOV3, MCF7 and T47D which are having
hormonal receptor positive. MCF7 and T47D
are cell lines from human breast cancer that
have estrogen receptor positive. SKOV3 is
human ovarian cancer cell lines which is also
having estrogen receptor positive. PC3 is
human prostate cancer cell-lines with
androgen hormone receptor positive. Their
activity on cell-lines presented by IC50 value,
which is means the effective
dose/concentration to inhibit 50% of cell
growth.
On PC3 cells, ethyl acetate phase of
TFG could inhibit growth of cell more
effective (IC
50
= 66.24 ppm) comparing with
methanolic and ethanolic extract. Prostate
cancer is a form of cancer that develops in the
prostate, a gland in the male reproductive
system. Most prostate cancer are slow
growing, however, there are cases of
aggressive prostate cancers.
On breast cancer cell-line, MCF7
and T47D, also the most effective
phase/extract is ethyl acetate phase of TFG
with IC
50
41.81ppm (on MCF7) and IC
50

58.63 ppm (on T47D). Breast cancer
(malignant breast neoplasm) is cancer
originating from breast tissue, most
commonly from the inner lining of milk ducts
or the lobules that supply the ducts with milk.
Cancer originating from ducts is known as
ductal carcinomas those originating from
lobules are known as lobular carcinomas.
Nowadays, breast cancer treatment,
especially with estrogen receptor positive,
commonly use tamoxifen, one of SERM
group. But the risk of taking this medicine in
long time is also high. Many research were
done to search another substance in this
group, especially from phytoestrogen, which
is have estrogenic effect, but effective to treat
breast cancer with estrogen receptor positive.
Interestingly, Asian women who consume a
soy-rich diet (the most famous phytoestrogen)
have about a 6-fold lower risk of developing
breast cancer than their Western counterparts
(Key et al., 1990), and population-based
studies have suggested that consumption of a
phytoestrogen-rich diet is protective against
prostate and bowel cancer, as well as
cardiovascular disease. Hence, it is possible
that these phytoestrogens function as
chemopreventive agents.
On ovarian cells, methanolic extract
of TFG more active than ethanolic extract and
its ethylacetate phase. Ovarian cancer is the
cancer that forms in tissues of the ovary (one
of a pair female reproductive glands in which
the ova, or egg, are formed). Most ovarian
cancers are either ovarian epithelial
carcinomas (cancer that begins in the cells on
the surface of the ovary) or malignant germ
cell tumors (cancer that begins in egg cells).
Predicted substances of TFG that
have estrogenic effect are some sapogenin
steroids, such as diosgenin, tigogenin and
gitogenin. These substances could bind with
estrogen receptor in the cells and compete the
inner body estrogen to induce the
proliferation. SERM works by blocks
estrogen action in breast cell, but can activate
estrogens action in other normal cells, such
as bone, liver and uterine cells. These data
can be used for further investigation to screen
the most active substance of TFG, to find new
SERM from phytoestrogen.

CONCLUSION
Results showed that ethyl acetate
fraction gives the lowest IC
50
than another
extracts. IC
50
for PC3 is 66.24 ppm, IC
50
for
MCF7 is 41.81 ppm, IC
50
for T47D is 58.63
ppm. These datas can be used for further
research to isolate the active compound from
TFG.
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